This application relates to a method and test kit for the detection of microorganisms in a liquid sample. The invention relies upon the ability of microbial dehydrogenase enzymes to reduce certain materials to produce visibly colored and therefore detectable products and capitalizes on this ability to produce simple and reliable kits to detect microorganisms such as bacteria generally or just gram negative bacteria.
It has long been recognized that dehydrogenases in living cells can be localized by histochemical methods within leucocytes and histological tissue sections by replacing the natural hydrogen acceptor with a reducible material, such as nitroblue tetrazolium as a ditetrazolium chloride, having a colored, water insoluble reduction product. Michel, G. et al. Methods in Enzymatic Analyses, Vol. 1, pp. 197-232 (1983). A similar approach has been used to detect living bacteria by observing whether a color change occurs upon the addition of a material such as triphenyl tetrazolium chloride (TTC), methylthiazolyldiphenyl tetrazolium bromide (MTT), iodonitrotetrazolium (INT) or neotetrazolium chloride (NTC), all of which turn red upon reduction, or blue tetrazolium (BT) or nitroblue tetrazolium (NBT) which turn blue upon reduction. Histological and Histochemical Methods, Theory and Practice, 2d. Ed. Chapter 16, p. 258 (1990).
The ability of living cells to reduce tetrazolium salts has formed the basis of a research tool to investigate the effects of various materials on bacterial respiration, i.e., oxygen uptake. In this regard, it has been reported that anionic detergents such as Tergitol-7 selectively inhibit the respiration of Gram-negative bacteria and consequently the reduction of tetrazolium salts to colored products. Cationic detergents inhibit respiration of both Gram-negative and Gram-positive bacteria, whereas neutral, non-ionic detergents are reported to have no effect on respiration. Baker et al., J. Exp. Med. 73, pp. 249-271 (1941). Dehydrogenase activity appears to be inhibited as well. Dakay et al., Zentralblatt Bakt. Hyg. I. Abt. Orig. B. 174, pp. 121-124 (1981).
The differential effects of detergents on dehydrogenase activity has been used as the basis for identifying bacterial types in liquid samples. European Patent Application 107594. It is an object the present invention to further exploit this ability of living bacteria and provide a method and test kit for simple and rapid determination of total microorganisms and Gram-negative bacteria.